The present invention relates generally to a method of isolating RNA-binding proteins. Particular modified bacterial host cells that are used in these methods are also provided.
RNA-binding proteins (RNA-BPs) play a key role in a variety of post-transcriptional regulatory processes, including RNA processing, nucleocytoplasmic transport, translation, and mRNA decay. Moreover, a burgeoning body of evidence has implicated a number of RNA-BPs in the genetic etiology of human diseases such as fragile X mental retardation (Ashley et al. (1993) Science 262:563-566), paraneoplastic neurologic disorders (Musunuru et al. (2001) Annu. Rev. Neurosci. 24:239-262), and spinal muscular atrophy (Fischer et al. (1997) Cell 90:023-1029), as well as in many microbial infections, including AIDS (Cullen (1998) Cell 93:685-692) and influenza (Garcia-Sastre 2001) Virology 279:75-384).
An essential step in elucidating the regulatory mechanism of a given RNA element is to identify and characterize the protein(s) that binds to this element and mediates its effect. Characterization of such an RNA-BP is most readily accomplished by cloning its cDNA. Traditionally, this has been achieved by protein purification, peptide microsequencing, and cDNA amplification with PCR primers designed on the basis of the amino acid sequence information. This labor-intensive strategy requires an enormous effort and can be particularly difficult in the case of RNA-BPs that are present at a relatively low cellular concentration. Some genetic screening methods have been developed to facilitate the cloning of RNA-BP cDNAs, most notably the yeast three-hybrid system (SenGupta et al. (1996) Proc. Natl. Acad. Sci. USA 93:8496-8501; Putz et al. (1996) Nucleic Acids Res. 24:4838-4840) and plaque-lift analysis (Qian et al. (1993) Anal. Biochem. 212: 47-554; Sagesser et al. (1997) Nucleic Acids Res. 25:3816-3822). Both techniques have allowed the cloning of previously unidentified RNA-BPs (Wang et al. (1996) Genes Dev. 10:3028-3040; Martin et al. (1997) EMBO J. 16:769-778; Zhang et al. (1997) Nature 390:477484; Denti et al. (2000) Nucleic Acids Res. 28:1045-1052).
However, several limitations may interfere with the general applicability of these procedures. For example, when using the three-hybrid system, the RNA-protein interaction takes place in vivo, where it can be influenced by a multitude of cellular parameters. False-positive clones frequently predominate, and these must be eliminated by additional time-consuming screening steps. For their part, plaque-lift assays are often severely hampered by inefficient or nonspecific binding of the radiolabeled RNA probe to the filter-bound plaques.
There is a need to design methodology that can be used to isolate new RNA-binding proteins. In addition, there is a need to construct particular modified host cells useful in performing such methodology.
The present invention provides a general in vitro method for isolating proteins that bind to specific RNA regulatory elements. The method employs a lyt-lys bacteriophage display library and a host bacterial cell that has been modified so as to release minimal-to-no RNase activity in the corresponding phage lysate. In a particular embodiment, the host cell has been modified to lack one particular ribonuclease activity. In a preferred embodiment, the host cell has been modified to lack one particular ribonuclease activity by deleting a particular gene that encodes the ribonuclease responsible for that activity.
In a particular embodiment, that ribonuclease activity is one that is otherwise responsible for the majority (or preferably all) of the RNase activity in the phage lysate. In another embodiment the particular ribonuclease activity is due to a ribonuclease that is not required for host cell viability. In a preferred embodiment, the ribonuclease activity is both one (i) that is otherwise responsible for the majority (or all) of the RNase activity in the phage lysate, and (ii) that ribonuclease activity is due to a ribonuclease that is not required for host cell viability.
In one embodiment, the bacterial host is a modified Gram negative bacterium. In a more specific embodiment, the modified bacterium is an enteric bacterium (i.e., belonging to the Enterobacteriaceae Family). In one such embodiment the modified bacterial host is Salmonella. In another such embodiment the modified bacterial host is Shigella. In still another embodiment, the modified bacterial host is Yersinia. In yet another embodiment, the modified bacterial host is Enterobacter. In still another embodiment, the modified bacterial host is Klebsiella. In a preferred embodiment, the modified bacterial host is Escherichia coli. Preferably the modified host bacterial cell had been modified to lack a periplasmic ribonuclease.
In a particular embodiment exemplified below, a specific E. coli strain (BLT5615, Novagen) is modified so that it lacks the gene for a major nonspecific E. coli ribonuclease, Rnase I (amino acid sequence of SEQ ID NO:1). This novel strain, has the T genotype Fxe2x88x92 rna ompT gal hsdSB (rBxe2x88x92mBxe2x88x92) dcm lac pAR5615 (ampR), herein known as RNA5615, and is part of the present invention.
By genetically deleting the RNase I (rna) gene from strain BLT5615, a variant E. coli strain (RNA5615) as been created which, when used with the T7 SELECT DISPLAY SYSTEM (U.S. Pat. No. 5,223,409 Issued Jun. 29, 1993 and U.S. Pat. No. 5,403,484 Issued Apr. 4, 1995, the contents of which are herein specifically incorporated by reference in their entireties) produces crude phage lysates that do not degrade RNA, even after prolonged incubation in the presence of magnesium ions. Consequently, phages that bind to an interesting RNA target can be isolated from crude phage lysates without first having to be purified by additional steps such as by sucrose gradient centrifugation.
The present invention therefore provides methods of identifying nucleic acids that encode RNA-binding proteins that bind to specific RNA regulatory elements. Once these nucleic acids are identified, the corresponding RNA binding proteins can be readily determined. One such method comprises inserting a cDNA into a lyt-lys phage cloning vector encoding the major capsid protein of the lyt-lys phage such that a heterologous protein encoded by the cDNA is expressed as a fusion protein with capsid protein when the cloning vector is packaged in a phage capsid. Further, the fusion protein is constructed such that the heterologous protein is accessible to the buffer and/or solvent when the resulting capsid is in solution. This ensures that the heterologous protein is accessible for binding an external ribonucleic acid (see FIG. 1).
The lyt-lys cloning vector is then packaged in a phage capsid in a host bacterial cell under conditions in which the phage capsid lyses the host cell after being packaged, thus forming a lysate. A host bacterial cell is used which previously had been modified so as to release minimal-to-no RNase activity in the corresponding phage lysate. This relatively RNase-free lysate is then contacted with a ribonucleotide that comprises a specific RNA regulatory element. When a phage capsid comprises a nucleic acid that encodes a heterologous protein that binds the specific RNA regulatory element, the phage capsid binds to the ribonucleotide forming an RNAapsid complex. The ribonucleotides and the RNA-capsid complexes are next separated from the lysate. The isolated capsids can then be used to infect fresh host bacterial cells leading to the formation of a fresh lysate. The fresh lysate can then be contacted with fresh ribonucleotides comprising the specific RNA regulatory element. Indeed, this in vitro selection process is preferably iterative. In any case, the nucleic acid encoding the heterologous protein of the phage capsid of the final RNA-capsid complex isolated is identified. This nucleic acid will encode an RNA-binding protein that binds to the specific RNA regulatory element.
In one particular embodiment of this method, the lyt-lys bacteriophage is T7, the cloning vector is a T7 cloning vector, the major capsid protein is T7 capsid protein 10B, and the host cell is E. coli RNA5615 having the genotype Fxe2x88x92 rna ompT gal hsdSB (rBxe2x88x92mBxe2x88x92) dcm lac pAR5615 (ampR).
The present invention further provides kits for identifying a nucleic acid that encodes an RNA-binding protein that binds to a specific RNA regulatory element. One such kit comprises a T7 cloning vector encoding T7 capsid protein 10B and an E.coli cell having the genotype Fxe2x88x92 rna ompT gal hsdSB (rBxe2x88x92mBxe2x88x92) dcm lac pAR5615 (ampR). In a preferred embodiment a streptavidin-coated paramagnetic bead is also included. In a particular embodiment the kit further comprises a protocol for performing the identification.
The present invention to provide an in vitro method for isolating RNA-binding proteins through the use of a phage display library and either undegraded or minimally degraded ribonucleic acids that comprise RNA regulatory elements or protein binding portions thereof.
Further, the present invention provides a bacterial host cell that has been modified so as to not encode an active periplasmic RNase that degrades RNA regulatory elements or protein binding portions thereof.
These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.